UNIVERSITY  OF  CALIFORNIA   PUBLICATIONS 

IN 

AGRICULTURAL    SCIENCES 

Vol.  3,  No.  5,  pp.  63-102  November  30,  1917 


TOXIC     AND     ANTAGONISTIC     EFFECTS     OF 

SALTS  ON  WINE  YEAST  (SACCHAROMYCES 

ELLIPSOIDEUS) 


BY 

S.  K.  MITBA 


CONTENTS 

PAGE 

Introduction    64 

Acknowledgments    65 

Method  of  experimentation 65 

Choice  of  solution 66 

Method  of  determining  the  activity  of  yeast  67 

Salts  used 68 

Single  salts — Toxic   effects  68 

Combinations — Antagonistic  effects  68 

Binary  combinations  68 

Ternary  combinations  68 

Experimentation  with  single  salts — Toxic  effects  68 

Series  I.     Potassium  chloride  69 

Series  II.     Magnesium   chloride   71 

Series  III.     Calcium  chloride  72 

Series  IV.     Sodium  chloride  74 

Series  V.    Effect  of  the  toxicity  of  salts  on  microscopical  appearance  of 

yeast  cells 75 

Experimentation  with  combinations  of  salts — Antagonistic  Effects  80 

Series  VI.    Antagonism  between  Magnesium  Chloride  and  Calcium  Chloride  81 

(a)  Plants 83 

(ft)   Animals    83 

(c)   Bacteria 84 

Series  VII.    Antagonism  between  Potassium  Chloride  and  Calcium  Chloride  84 

(a)   Plants 86 

(&)   Animals 87 

(c)   Bacteria 87 

Series  VIII.  Antagonism  between  Magnesium  Chloride  and  Sodium  Chloride  87 

(a)   Plants   89 

(6)   Animals    89 

(c)   Bacteria  90 


64  University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 

PAGE 

Series  IX.     Antagonism  between  Potassium  Chloride  and  Sodium  Chloride  90 

(a)  Plants 90 

(&)   Animals    90 

(c)   Bacteria    92 

Series  X.  Antagonism  between  Potassium  Chloride  and  Magnesium  Chloride  92 

(a)   Plants   94 

(6)   Animals    94 

(c)   Bacteria 94 

Series  XL     Antagonism  between  Calcium  Chloride  and  Sodium  Chloride  ....  94 

(a)   Plants   95 

(&)   Animals    96 

(c)   Bacteria    96 

Eelative  antagonisms  of  various  combinations  97 

Summary    99 

Part  A.     Toxic  effects  of  single  salts  99 

Part  B.     Antagonistic  effects  of  combinations  of  salts  99 

Literature  cited  101 

Part  A.     Toxic  effects  of  single  salts  101 

Part  B.    Antagonistic  effects  of  combinations  of  salts  101 


Introduction 

Most  of  the  published  studies  of  wine  yeast  deal  with  its  activities 
as  related  to  wine  making.  Its  botanical  characteristics  and  still  less 
its  fundamental  physiological  reactions  have  apparently  received  little 
attention.  '  Among  the  most  important  and  interesting  investigations 
of  higher  plants,  bacteria  and  animals  in  recent  years  have  been  studies 
of  the  effects  of  various  single  salts  and  various  combinations  of  salts 
on  the  physiology  of  these  organisms.  For  example,  it  has  been  found 
by  Osterhout6'  7  that  practically  all  of  the  simple  salts,  such  as 
sodium  chloride,  potassium  chloride,  calcium  chloride,  etc.,  have  a 
decided  toxic  action  upon  the  plant  when  it  is  subjected  to  the  action 
of  a  single  pure  salt.  Further,  if  certain  combinations  of  two  or  more 
salts  were  used  in  certain  ratios  the  toxicity  was  reduced.  This 
reduction  of  toxicity  is  commonly  termed  antagonism  between  the 
salts  used.  It  was  found  also  that  a  combination  of  all  the  salts  in 
the  ratios  in  which  they  occur  in  the  soil  solutions  or  in  other  solutions 
to  which  the  plant  is  accustomed  afford  the  best  conditions  for  growth. 
Such  a  combination  is  spoken  of  as  a  physiologically  balanced  solution. 

Loeb  working  with  marine  animals  and  C.  B.  Lipman  with  soil 
and  other  bacteria  obtained  results  similar  to  those  obtained  by  Oster- 
hout with  the  higher  plants.  As  was  to  be  expected,  the  reaction  of 
animals  to  the  salts  was  not  identical  with  that  of  bacteria,  nor  does 
either  reaction  follow  the  behavior  of  the  higher  plants  closely.     In 


1917]         Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast  65 

general,  however,  the  results  with  all  three  classes  were  of  the  same 
kind;  that  is,  single  salts,  hitherto  considered  non-toxic,  were  found 
to  be  toxic  to  organisms,  while  various  combinations  of  these  salts 
showed  antagonism  or  reduction  of  toxicity  in  the  presence  of  each 
other.  In  accordance  with  these  facts,  a  physiologically  balanced 
solution  can  be  made  by  using  proper  concentrations  and  proportions 
of  the  various  salts  found  in  the  solutions  to  which  the  organism  is 
accustomed. 

So  far  as  the  writer  could  ascertain,  no  one  has  investigated  the 
behavior  of  yeast  in  this  respect.  Results  of  investigations  of  the 
effects  of  the  heavy  metallic  salts,  such  as  mercuric  chloride,  silver 
nitrate,  etc.,  on  yeast  have  been  published  (Bokarny12),  but  nothing 
has  appeared  upon  the  toxic  and  antagonistic  effects  of  such  salts  as 
sodium  chloride,  potassium  chloride,  calcium  chloride,  and  magnesium 
chloride.  This  field  therefore  seemed  especially  inviting,  and  it  was 
with  the  idea  of  studying  the  fundamental  relations  existing  between 
yeast  and  the  chlorides  that  the  writer  undertook  the  work  summarized 
in  the  following  pages. 

Acknowledgments 

The  experiments  on  which  this  paper  is  based  were  carried  out 
under  the  general  supervision  of  Professor  W.  V.  Cruess,  and  I  am 
indebted  to  Professor  P.  T.  Bioletti  for  suggestions  and  critical  read- 
ing of  the  manuscript. 

Method  of  Experimentation 

In  the  selection  of  a  yeast  for  my  investigation  I  was  led  to  use 
the  wine  yeast,  Saccharomyces  ellipsoideus,  by  the  fact  that  it  is  one 
of  the  most  useful  of  all  yeasts  and  is  universally  used  in  wine  making. 
It  is  also  one  of  the  most  vigorous,  is  easy  to  grow,  and  gives  definite 
results  in  a  few  days. 

The  particular  yeast,  no.  66,  used  in  this  experiment,  was  isolated 
by  William  V.  Cruess  from  one  of  the  wineries  in  northern  California. 
It  has  been  found  by  repeated  trials  that  specimens  of  Saccharomyces 
ellipsoideus  collected  from  different  sources  are  not  identical  as  to 
their  physiological  characters  in  every  respect,  and  so  do  not  respond 
in  different  salt  solutions  in  the  same  way.  An  experiment  showing 
this  will  be  described  later. 

Although  work  on  this  line  may  not  have  immediate  practical 


66  University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 

value  to  the  zymologist,  it  is  of  considerable  scientific  interest.  It  is 
with  this  thought  that  these  experiments  have  been  carried  out  in  the 
Laboratory  of  Zymology.* 

The  salts  tested  in  these  experiments  were  the  chlorides  of  potas- 
sium, magnesium,  calcium  and  sodium,  each  being  taken  up  separately. 
The  reason  for  choosing  these  chlorides  was  that  their  metallic  ions 
(cations)  are  those  most  abundant  in  the  ash  of  grape  juice.  Besides, 
Loeb1  and  Lipman11  have  shown  that  the  positive  ions  of  these  salts 
have  the  most  effect,  while  their  negative  ions  (anions)  have  the  least. 
Owing  to  the  fact  that  the  effect  of  the  chlorine  ion  is  uniform  in 
all  cases,  the  metallic  ions  show  their  characteristic  effects  on  the  yeast 
culture  very  clearly. 

Choice  of  Solution. — It  has  been  shown  by  Loeb  with  marine  ani- 
mals (Fundulus) ,  by  Osterhout  with  higher  plants  (wheat),  and  by 
Lipman  with  soil  bacteria  (Bacillus  subtilis)  that,  for  the  growth  of 
living  organisms,  a  nutrient  solution  must  be  physiologically  balanced. 
In  order  to  grow  the  yeast  in  a  medium  whose  constituents  were 
known  both  in  quality  and  quantity,  it  was  necessary  to  prepare 
a  nutrient  solution  from  pure  materials.  Such  a  solution  must  con- 
tain an  adequate  amount  of  nitrogen  and  phosphorus  in  order  that 
the  yeast  may  grow  rapidly.  For  this  purpose  a  number  of  sub- 
stances were  tried,  such  as  Witte's  peptone,  asparagin,  urea,  and 
ammonium  phosphate,  in  different  concentrations,  with  pure  cane 
sugar  or  pure  dextrose.  Witte's  peptone  proved  to  be  impure,  being 
very  high  in  ash,  and  the  others  did  not  give  satisfactory  results. 
As  dextrose  is  not  easily  available  in  the  market  at  present,  pure  cane 
sugar  had  to  be  used  as  a  carbohydrate  food  and  as  a  source  of  fer- 
mentable material. 

Later  a  synthetic  solution  was  made  with  hydrolizecl  pure  cane 
sugar,  phosphoric  acid,  and  ammonia,  which  was  suitable  for  the 
growth  of  yeast  for  experimental  purposes.  Although  this  synthetic 
solution  produces  a  slower  rate  of  growth  than  grape  juice,  which  is 
a  perfect  physiologically  balanced  solution  for  yeast  fermentation,  it 
gives  sufficient  growth  for  experimental  work.  To  make  it,  a  50  per 
cent  pure  cane  sugar  syrup  was  made  with  distilled  water  and  a 
measured  amount  (1  gram  per  100  c.c.  of  syrup)  of  phosphoric  acid 
added.  This  syrup  was  hydrolized  by  boiling  for  one-half  hour  on 
a  slow  fire,  and  was  then  neutralized  with  dilute  ammonium  hydroxide. 
Litmus  solution  was  used  to  test  the  neutrality  of  the  syrup.     The 


*  These  experiments  were  earried  out  under  the  general  supervision  of  William 
Y.  Cruess. 


1917]        Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast  f>7 

ammonium  phosphate  that  eventually  formed  furnished  both  the 
nitrogen  and  phosphorus  needed  by  the  yeast.  As  yeast  eells  grow 
easily  in  moderately  acid,  but  not  in  alkaline  solutions,  the  syrup 
was  left  slightly  acid,  being  tested  by  titration  with  N/10  solution  of 
sodium  hydroxide.  To  facilitate  the  work,  the  syrup  was  boiled  down 
to  65°Balling  and  put  into  a  corked  bottle.  From  this  concentrated 
synthetic  solution  a  measured  quantity  was  drawn  off  and  diluted  with 
distilled  water  to  5°Bal.  for  use  in  the  cultures. 

Method  of  Determining  the  Activity  of  the  Yeast. — The  experi- 
ments were  carried  on  in  a  series  of  200  c.c.  Erlenmyer  flasks.  To 
each  flask  the  weighed  amount  of  salt  was  added  and  100  c.c.  of  the 
diluted  synthetic  solution  placed  in  the  flask  by  means  of  a  100  c.c. 
pipette. 

The  salts  were  weighed  according  to  their  respective  molecular 
concentrations.  The  flasks  as  soon  as  filled  were  plugged  with  cotton 
and  sterilized.  After  they  had  cooled  down  to  the  room  temperature 
they  were  inoculated  with  the  yeast  from  a  new  culture.  For  this 
purpose  the  new  culture  was  prepared  in  a  200  c.c.  Erlenmyer  flask 
containing  100  c.c.  of  the  synthetic  solution.  The  yeast  thus  became 
habituated  to  this  solution  and  therefore  grew  rapidly  and  uniformly 
in  the  flasks.  The  new  culture  was  transferred  from  a  mother  culture 
in  grape  juice  and  put  into  the  incubator  for  forty-eight  hours  at 
28° C.  At  the  end  of  this  period  the  flasks  containing  the  salts  were 
inoculated  with  one  cubic  centimeter  of  the  new  culture.  After 
inoculation,  the  flasks  were  put  into  the  incubator,  which  was  kept 
at  an  approximately  even  temperature  of  28° C.  during  the  entire 
experiment. 

As  the  alcoholic  fermentation  in  the  synthetic  solution  was  not 
rapid  enough  to  serve  as  a  criterion,  the  multiplication  of  the  cells 
was  taken  as  the  measure  of  the  activity  of  the  yeast.  Accordingly 
a  microscopical  count  was  made  every  forty-eight  hours  with  a  cali- 
brated microscope.  Five  counts  were  made  in  each  experiment,  during 
a  period  of  about  twelve  days.  In  every  case  two  blanks,  with  no 
added  salts,  were  made  up,  and  the  tables  given  represent  the  average 
of  two  sets  of  duplicate  experiments,  except  in  the  cases  of  potassium 
chloride  and  magnesium  chloride,  where  the  results  were  so  close  that 
only  the  first  set  of  duplicates  was  used.  It  may  be  added  that  the 
incubator  did  not  keep  exactly  the  same  temperature  throughout  the 
experiments,  but  ranged  from  27 °C.  to  29 °C.  This  difference  of  2°C., 
however,  did  not  interfere  appreciably  with  the  uniformity  of  growth 


68  University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 

and  the   check  flasks   controlled   any   slight   variation   it   may  have 
caused. 

Salts  Used. — During  February,  1916,  a  series  of  experiments  in 
two  parts  was  planned.  The  first  concerned  the  toxicity  of  the  single 
salts  and  the  second  the  antagonistic  effects  of  all  their  binary  and 
ternary  combinations,  as  follows: 

I.  Toxicity  of  the  Single  Salts 

1.  KC1  3.  CaCl2 

2.  MgCL  4.  NaCl 

II.  Antagonistic  Effects  of  Combinations 
A.  Binary  Combinations 

1.  MgCl2  +  CaCL  4.  KC1  +  NaCl 

2.  KC1  +  CaCL  5.  KC1  +  MgCL 

3.  MgCl2  +  NaCl  6.  CaCL  +  NaCl 

B.  Ternary  Combinations 

1.  NaCl  +  KC1  +  CaCL  3.  NaCl  +  MgCL  +  CaCL 

2.  NaCl  +  KC1  +  MgCL  4.  KC1  +  CaCL  +  MgCL 


A.  Experiments  With  Single  Salts — Toxic  Effects 

In  all  cases  chemically  pure  salts  (Baker's  analyzed)  were  used. 
Each  amount  of  the  single  salts  was  carefully  weighed  and  put  into 
the  flasks,  except  .001M  and  .01M,  which  were  added  as  solutions  of 
known  strength  according  to  molecular  weights.  The  following  pro- 
portions were  taken: 

1.  KCl  —  .001M  to  2.2M*  Molecular 

2.  MgCL  —  .001M  to  1.2M 

3.  CaCl2  —  .001M  to  .7M 

4.  NaCl  —  .001M  to  .2M 

All  of  these  solutions  were  clear  except  the  calcium  chloride,  which 
gave  an  appreciable  amount  of  coagulated  precipitate  of  calcium  phos- 
phate with  the  phosphorus  of  the  synthetic  solution.  This,  however, 
did  not  interfere  with  the  experiment,  as  the  precipitate  disappeared 
with  the  growth  of  the  yeast  and  the  solution  finally  became  almost 
clear.  The  tables  and  curves  given  in  each  case  show  the  growth  of 
yeast  at  every  forty-eight  hours  in  the  different  molecular  concen- 


*  M  represents  the  degree  of  concentration  in  a  solution  which  contains  one 
gram  molecule  of  the  substance  in  one  litre  of  solution. 


1917]        Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast  69 

trations  of  each  salt  as  indicated  above.  In  the  microscopical  count 
one  million  yeast  cells  per  cubic  centimeter  was  taken  as  an  appreci- 
able number ;  below  that  it  was  not  considered  that  any  appreciable 
growth  had  taken  place. 


SERIES  I— POTASSIUM  CHLORIDE 

Thirty  200  c.c.  Erlenmyer  flasks  were  arranged  in  duplicate,  in- 
cluding two  blanks.  The  first  pair  had  no  salt  added  and  was  used 
as  a  check.  The  second  pair  had  .001M  KC1  and  the  third  .01M  KC1, 
and  so  on  to  the  last  pair,  which  had  2.2M  KC1,  as  shown  in  the  table 
below.  Then,  with  a  100  c.c.  pipette  one  hundred  cubic  centimeters 
of  the  diluted  synthetic  solution  (5°Bal.)  were  put  into  each  flask. 
The  flasks  were  plugged  with  cotton,  sterilized,  and  inoculated  with 
yeast,  as  stated  before,  and  put  into  the  incubator,  and  counted  every 
forty-eight  hours.  The  results  are  shown  in  table  1  and  the  curves 
in  figure  1. 

The  curves  have  been  plotted  from  the  results  of  every  forty-eight 
hours'  growth,  taking  the  various  concentrations  of  potassium  chloride 
as  abscissae  and  the  number  of  yeast  cells,  counted  in  millions,  as 
ordinates  (fig.  1).  Following  the  table  and  the  curves,  it  is  evident 
at  a  glance  that  potassium  chloride  up  to  the  concentration  of  .2M 
accelerates  the  multiplication  of  the  yeast.  Bej^ond  this  it  becomes 
gradually  more  and  more  toxic  until  at  2.2M  the  yeast  cells  entirely 
cease  to  multiply.  Both  Magowan10  and  Lipman,11  especially  the 
former,  found  a  strong  resemblance  between  potassium  chloride  and 
sodium  chloride  in  their  action  on  wheat  and  on  Bacillus  subtilis. 
The  yeast  shows  physiological  characteristics  differing  from  those  of 
either  the  bacteria  or  the  wheat. 

Lipman11  found  sodium  chloride  the  least  toxic  to  Bacillus  subtilis 
and  potassium  chloride  second.  Magowan,10  with  wheat,  found  this 
position  reversed,  the  potassium  chloride  being  less  toxic.  Both  ob- 
servers found  that  these  salts  were  very  similar  in  their  degree  of 
toxicity.  To  yeast  sodium  chloride  is  the  most  toxic  of  the  four  salts 
and  potassium  chloride  the  least.  Ostwald4  in  experimenting  with 
animals  (Grammarus)  found  potassium  chloride  the  most  toxic,  and 
Loeb  's  work2' 3  with  Fundulus  corroborates  this  to  a  certain  extent. 
The  reaction  of  yeast,  therefore,  differs  from  that  of  bacteria  of  the 
higher  plants  or  of  animals. 


70 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.  3 


Table  1 — Toxic  Effect  of  KCl  on  Saccharomyces  ellipsoideus 

M.KCl  48  hrs.                    96  hrs.                144  hrs.  192  hrs.                240  hrs. 

.00  1,695,000    7,237,000   11,636,000  12,892,000  15,835,000 

.001  3,325,000   10,786,000   15,400,000  16,217,000  19,670,000 

.01  7,797,000   15,600,000   18,905,000  21,479,000  23,568,000 

.1  7,262,000   20,558,000   24,208,000  24,608,000  27,915,000 

.2  5,650,000   18,493,000   29,689,000  28,783,000  30,541,000 

.4  5,425,000   20,227,000   25,006,000  27,560,000  28,914,000 

.6  1,130,000    8,535,000   20,928,000  22,802,000  23,802,000 

.8  809,000    5,298,000   17,670,000  19,508,000  20,982,000 

1.0  226,000    6,787,000   15,205,000  17,986,000  18,453,000 

1.2    1,102,000    8,986,000  16,207,000  16,951,000 

1.4    452,000    8,765,000  11,584,000  12,882,000 

1.6    3,204,000  5,794,000  8,232,000 

1.8    904,000  1,243,000    6,252,000 

2.0    904,000    2,051,000 

2.2  226,000 


A         .6         .8         1.0       1.2 
Concentration  of  salt 


Fig.  1. — The  ordinates  represent  millions  of  yeast  cells  and  the  abscissae, 
the  various  concentrations  of  KCl.  The  ordinates  at  0  represent  the  number 
of  yeast  cells  in  the  check  cultures. 


1917]         Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast 


71 


SERIES  II— MAGNESIUM  CHLORIDE 

The  same  method  of  procedure  was  used  with  MgCl2  as  with  KC1. 
The  results  are  shown  in  Table  2. 


Table  2- 

-Toxic  Effect  of  MgCl2 

on  S.  ellipsoideus 

M.  MgClo 

48  hrs. 

96  hrs. 

144  hrs. 

192  hrs. 

240  hrs. 

.00 

2,412,000 

8,108,000 

12,205,000 

16,837,000 

17,202,000 

.001 

4,972,000 

10,753,000 

13,673,000 

18,871,000 

19,775,000 

.01 

8,751,000 

15,798,000 

18,703,000 

19,588,000 

20,374,000 

.1 

3,482,000 

12,227,000 

20,521,000 

25,013,000 

26,501,000 

.2 

2,356,000 

7,204,000 

11,342,000 

15,530,000 

18,617,000 

.4 

1,695,000 

5,400,000 

9,504,000 

12,837,000 

17,413,000 

.6 

989,000 

2,157,000 

7,332,000 

11,253,000 

11,905,000 

.8 

226,000 

1,130,000 

4,294,000 

6,943,000 

8,436,000 

1.0 

226,000 

1,875,000 

2,712,000 

2,904,000 

1.2 

226,000 

.01        .1         .2  .4 

Concentration  of  salt 


Fig.  2. — The  ordinates  represent  the  number  of  yeast  cells  in  millions  and 
the  abscissae,  the  concentration  of  magnesium  chloride.  The  ordinate  at  0 
represents  the  number  of  yeast  cells  in  the  check  cultures. 


72  University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 

From  both  table  2  and  the  curves  in  figure  2,  it  is  evident  that 
magnesium  chloride  is  more  toxic  than  potassium  chloride.  Up  to 
the  concentration  of  .1M,  it  is  favorable  to  the  growth  of  yeast,  but 
beyond  this  it  becomes  more  and  more  toxic  until  at  1.2M  concen- 
tration there  is  little  or  no  growth  at  all.  In  the  case  of  yeast,  mag- 
nesium chloride  and  calcium  chloride  show  less  similarity  than  that 
found  by  Lipman  with  soil  bacteria,  and  the  toxic  effect  of  magnesium 
chloride  is  nearer  to  that  of  calcium  chloride  than  to  that  of  potassium 
chloride.  Magnesium  chloride  is  not  so  toxic  to  yeast  as  Lipman 
found  it  with  Bacillus  subtilis.  In  the  case  of  yeast,  .7M  concen- 
tration of  calcium  chloride  altogether  inhibits  its  growth.  The  same 
concentration,  however,  of  magnesium  chloride  allows  an  appreciable 
number  of  yeast  cells  to  grow,  and  the  same  toxic  effect  as  that  of 
.7M  CaCL  is  not  attained  until  a  concentration  of  1.2M  MgCl2  is 
reached.  In  fact,  magnesium  chloride  stands  midway  between  the 
two  extremes  of  toxicity  of  these  four  salts,  namely,  the  more  toxic 
NaCl  and  CaCl2  and  the  less  toxic,  KC1. 

Loeb,1, 2  with  marine  organisms,  found  that  a  .5M  solution  of 
magnesium  chloride  inhibits  the  development  of  embryos  in  the  eggs 
of  Fundulus,  and  that  even  .125M  Ca(N03)2  is  toxic.  In  his  experi- 
ment with  soil  bacteria  Lipman11  has  met  with  about  the  same  result. 
He  found  that  .4M  MgCl2  inhibits  the  growth  of  Bacillus  subtilis, 
while  for  the  same  effect  on  yeast  a  concentration  of  1.0M  MgCl2  is 
needed.  But  Magowan10  has  shown  that  with  wheat  magnesium  chlor- 
ide is  the  most  toxic  of  all  the  four  salts ;  in  this  respect  yeast  resembles 
neither  the  animals,  nor  bacteria,  nor  the  higher  plants. 

It  must  be  noted  that  the  magnesium  chloride  used  in  all  the 
experiments  was  MgCl2.6H20,  as  this  is  less  hygroscopic  than  the 
same  salt  having  two  molecules  less  of  water  (MgCl2.4H20),  which 
is  difficult  to  weigh  accurately.  However,  magnesium  chloride  was 
found  more  toxic  than  potassium  chloride  and  more  favorable  than 
calcium  chloride,  which  is  directly  opposite  to  the  result  obtained 
with  higher  plants. 


SERIES  III— CALCIUM  CHLORIDE 

The  experiment  with  calcium  chloride  was  carried  on  in  the  same 
Avay.  From  both  table  3  and  the  curves  in  figure  3,  it  is  evident  that 
.01M  concentration  of  CaCl2  gives  the  highest  growth,  while  beyond 
this  favorable  concentration  CaCL  is  more  and  more  toxic.     In  its 


1917]         Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast 


7:i 


toxicity  to  yeast,  CaCl2  stands  second,  NaCl  being  the  most  toxic  of 
the  four  salts.  A  .5M  concentration  of  CaCL  allows  an  appreciable 
growth  of  yeast,  while  even  ,2M  NaCl  stops  all  growth. 

Table  3 — Toxic  Effect  of  CaCl2  on  S.  ellipsoideus 

M.  CaCI2      48  hrs.  96  hrs.                144  hrs.  192  hrs.                240  hrs. 

.00  2,599,000         8,136,000       11,752,000  13,108,000  16,336,000 

.001  3,051,000         9,989,000     •  12,656,000  13,965,000  17,751,000 

.01  3,503,000       11,050,000       13,926,000  15,885,000  19,251,000 

.1  226,000         6,034,000         8,468,000  10,904,000  16,674,000 

.2    '. 1,695,000    3,256,000  3,821,000  15,778,000 

.3    904,000    1,130,000  3,090,000  14,561,000 

.4    226,000     904,000  1,130,000    9,771,000 

.5    226,000  987,000    2,935,000 

.6    226,000     904,000 

.7 226,000 


19 

18 

16  ( 
8    14 

&   12 

/ 

y 

i 

\  tvJ 

•>  \  3s- 

w 

o 
c    10 

8  8< 

6 
4 

\     v\ 

/ 

i 

Vs  \ 
W 

\  l<5> 

V* 

\    ° 

\    *- 
\   ^ 

\    ^ 

s* 

< 
2 

\  c 

» 

0       .001        .1         .2         .3         .4         .5         .6         .7 
Concentration  of  salt 

Fig.  3. — The  ordinates  represent  the  number  of  yeast  cells  in  millions  and 
the  abscissae  the  concentration  of  CaCL.  The  ordinate  at  0  represents  the 
number  of  yeast  cells  in  the  check  cultures. 


The  work  of  other  investigators  with  regard  to  the  toxicity  of 
CaCl2  shows  a  general  agreement  with  the  results  obtained  by  Loeb 
with  Fundulus,3  Ostwald4  with  fresh  water,  Grammarus,  and  Lip- 
man11  with  soil  bacteria,  all  of  which  show  CaCL  to  be  extremely  toxic. 
An   exception   to  this  general   statement   is  found    in   the   work   of 


74 


University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 


Magowan,  who  showed  in  her  experiments  that,  in  the  case  of  wheat, 
CaCl2  is  the  least  toxic  of  the  four  salts.  Here  also  we  find  that 
yeast  exhibits  a  peculiar  physiological  character  which  does  not  agree 
with  either  of  the  above  divisions,  the  animals  or  the  plants.  Perhaps 
this  may  throw  some  light  on  the  relation  of  yeast  to  these  two  groups. 

SEKIES  IV— SODIUM  CHLORIDE 

As  NaCl  is  the  most  toxic  of  all  the  salts,  only  a  few  pairs  of  flasks 
were  taken,  from  .001M  to  .2M  concentration,  together  with  a  pair 
of  blanks.  The  experiment  was  carried  on  in  the  same  way  as  the 
others.  From  table  4  and  the  curves  in  figure  4,  it  is  evident  that 
NaCl  is  the  most  toxic  to  the  yeast.  Even  the  concentration  of  .01M 
NaCl  did  not  stimulate  the  growth  of  yeast,  as  it  did  with  the  other 
salts.  The  highest  growth  in  this  case  was  in  .001M,  and  beyond  that 
it  was  toxic. 


Table  4- 

-Toxic  Effect  of  NaCl 

on  8.  ellipsoideus 

M.NaCl 

48  hrs. 

96  hrs. 

144  hrs. 

192  hrs. 

240  hrs. 

.00 

2,424,000 

8,059,000 

9,267,000 

11,978,000 

15,142,000 

.001 

3,819,000 

9,605,000 

12,430,000 

12,995,000 

17,967,000 

.01 

3,164,000 

7,458,000 

10,283,000 

10,504,000 

13,060,000 

.1 

226,000 

452,000 

452,000 

452,000 

1,130,000 

.2 

226.000 

18 


16 


«    14 


■S    12 


10 


tf 

V 

^>7 

if* 

$^ 

0       .001       .01        .1         .2 
Concentration  of  salt 


Fig.  4. — The  ordinates  represent  the  number  of  yeast  cells  in  millions  and 
the  abscissae,  the  concentration  of  NaCl.  The  ordinate  at  0  represents  the 
number  of  yeast  cells  in  blank  cultures. 


1917]        Miira:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast  75 

NaCl  shows  a  directly  opposite  reaction  witli  yeast  from  that 
found  by  Lipman11  with  soil  bacteria.  Both  Loeb  and  Ostwald  found 
NaCl  to  be  toxic  for  animals,  but  less  so  than  we  have  found  with 
yeast.  The  toxicity  of  NaCl  to  animals  may  be  compared  with  the 
toxicity  of  NaCl2  to  yeast.  Loeb3  found  it  impossible  to  develop 
embryos  in  the  egg  of  Fundulus  at  .625M  NaCl.  Osterhout8' 9  found 
that  a  .375M  solution  of  sodium  chloride  is  fairly  toxic  to  marine 
plants.  Young  plants  of  a  fresh-water  alga,  Vaucheria  sessilis,  could 
not  live  at  a  .094M  concentration  of  sodium  chloride,  and  even  a 
concentration  of  .0001M  NaCl  was  found  to  be  toxic.  Magowan  has 
shown  that  sodium  chloride  is  very  toxic  to  wheat  seedlings  and  down 
to  .02M  the  root  hairs  did  not  grow  at  all.  The  relation  of  yeast  to 
plants  is  thus  to  a  certain  extent  shown  by  similar  physiological 
behavior. 

It  may  be  noted  here  that  the  experiments  with  yeasts  have  been 
conducted  on  the  same  general  principle  followed  by  previous  investi- 
gators with  animals,  plants,  and  bacteria.  The  number  of  yeast  cells 
was  taken  as  the  measure  of  multiplication  or  activity  and  was  de- 
termined by  a  microscopical  count  of  each  flask  every  forty-eight 
hours.  It  must  be  admitted  that  experimental  errors  may  occur  in 
counting,  but  as  the  numbers  were  taken  from  the  average  results  of 
two  sets  of  duplicates  it  does  not  interfere  with  the  validity  of  the 
final  result,  as  the  range  of  variation  between  the  results  of  these  two 
sets  of  duplicates  was  only  between  0  and  10  per  cent  calculated  from 
the  mean  variation. 


SERIES  V— EFFECT  OF  THE  TOXICITY   OF  SALTS  ON  THE 
MICROSCOPICAL  APPEARANCE  OF  YEAST  CELLS 

It  is  generally  known  that  all  salts  at  certain  concentrations  are 
more  or  less  toxic  to  living  organisms.  Yeast  shows  its  physiological 
condition  in  relation  to  various  salts  in  characteristic  ways.  It  is 
evident  from  the  above  experiments  that  in  this  respect  it  occupies 
a  place  between  the  animal  and  the  plant  kingdoms.  Although  yeast 
grows  normally  in  a  physiologically  balanced  solution,  for  which 
grape  juice  answers  in  every  way,  the  addition  of  a  small  amount  of 
a  favorable  salt,  as  potassium  chloride,  may  stimulate  the  growth  a 
great  deal.    This  is  of  some  practical  zalue  to  zymologists. 

Yeast  is  affected  very  remarkably  by  the  toxicity  of  salts  at  dif- 
ferent concentrations.     In  the  extreme  concentrations  it  apparently 


76  University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 

dissolves.  This  occurs  in  the  cultures  having  2.2M  KC1,  1.2M,  MgCl2, 
.7M  CaClo,  and  .2M  NaCl  respectively.  At  lower  concentrations  there 
is  a  degenerated  condition,  various  shapes  occurring,  as  shown  in 
figure  C.  Such  diseased  cells  show  a  heavy  black  membrane,  especially 
in  the  case  of  CaCl2  and  NaCl,  with  transparent  cell-illusions  or  black 
spots  within  the  cells.  Moreover,  they  vary  in  size.  This  variation 
in  size  occurs  also  with  KC1  and  MgCl2,  but  in  these  cases  the  yeast 
cells  are  larger  than  with  CaCl2  and  NaCl.  In  all  instances,  as  the 
concentration  of  salt  increases  beyond  the  favorable  degree  of  con- 
centration the  cells  become  smaller  and  smaller  until  finally,  in  the 
extreme  concentrations,  they  dissolve.  Table  5  (a.  b,  c,  d)  and  the 
curves  in  figure  5  {a,  b)  show  the  effect  on  the  size  of  yeast  cells  in 
different  salt  solutions. 

Table  5a — Effect  of  KCl  on  Size  of  Yeast  Cells  (S.  ellipsoideus) 


Concentration 

of  salt 

(M.KC1) 

Av.  length 

and  breadth  of 

yeast  cells 

in  Mu. 

Av.  volume 

yeast  cells 

calculated  from 

length  and  breadth 

.00 

4.7  X  4.6 

77 

.001 

5.4x5.4 

122 

.01 

6.7x6.7 

232 

.1 

6.7x6.7 

232 

.2 

6.7x6.7 

232 

.4 

6.6  x  6.6 

223 

.6 

6.6  x  6.6 

223 

.8 

5.8x5.8 

151 

1.0 

5.8x5.8 

151 

1.2 

5.8x5.8 

151 

1.4 

5.8x5.8 

151 

1.6 

4.9x4.9 

91 

1.8 

4.9  x  4.9 

91 

2.0 

3.3x3.3 

28 

2.2 

3.3x3.3 

28 

•Effect  of  MgCl2 

on  Size  of  Yeast  Cells   (S.  elli\ 

Concentration 

of  salt 

(M.MgClo) 

Av.  length 

and  breadth  of 

yeast  cells 

in  Mu. 

Av.  volume 

yeast  cells 

calculated  from 

length  and  breadth 

.00 

4.5x4.5 

71 

.001 

5.1x5.1 

103 

.01 

6.2  x  6.2 

185      • 

.1 

6.2x6.2 

185 

.2 

5.0x5.0 

98 

.4 

5.1x5.1 

103 

.6 

5.0x5.0 

98 

.8 

4.9x4.9 

91 

1.0 

4.4  x  4.4 

66 

1.2 

3.3  x  3.3 

28 

1917 J         Ultra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast 


77 


Table  5c— Effect  of  CaCl2  on  Size  of  Yeast  Cells  (S.  ellipsoideus) 


Concentration 

of  salt 

(M.CaClo) 

.00 

.001 

.01 

.1 

.2 
.3 
.4 
.5 
.6 
.7 


Av.  length 

and  breadth  of 

yeast  cells 

in  Mu. 

4.7x4.4 

Av.  volume 

of  yeast  cells 

calculated  from 

length  and  breadth 

70 

4.6x4.6 

75 

4.1x4.1 

53 

3.3x3.3 

28 

3.3x3.3 

28 

2.8x2.8 

17 

2.8x2.8 

17 

2.8x2.8 

17 

2.6x2.6 

14 

2.6x2.6 

14 

Table  5d — Effect  of  NaCl  on  Size  of  Yeast  Cells  (S.  eUipsoides) 


Concentration 

of  salt 

(M.NaCl) 

.00 

.001 

.01 

.1 

.2 


Av.  length 

ind  breadth  of 

of  yeast  cells 

in  Mu. 

Av.  volume 

of  yeast  cells 

calculated  from 

length  and  breadth 

5.1x4.8 

91 

5.0x4.4 

75 

4.4x3.3 

37 

4.1x3.3 

34 

2.6x2.6 

14 

.001       .01 


.8         1.0       1.2       1.4 

Concentration  of  salt 


1.6       1.8      2.0 


Fig.  5a. — Curves  showing  the  average  relative  volumes  of  yeast  cells  in 
various  concentrations  of  CaCL  and  MgCl2.  The  ordinates  represent  the  average 
volume  of  the  yeast  cells  and  the  abscissae,  the  concentrations  of  KC1  and  MgCL 
used.     The  ordinate  at  0  represents  the  volume  in  blank  cultures. 


78 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.  3 


200 

195 

100 

^ 

L* 

)^ 

\ 

,N 

) c 

0        .001       .01         .1  .2        .3         .4         .6         .7 

Concentration  of  salt 

Fig.  5b. — Curves  showing  the  average  relative  sizes  in  volumes  of  yeast  cells 
in  various  concentrations  of  KC1  and  MgCL.  The  orclinates  represent  the 
volume  of  the  yeast  cells  and  the  abscissae,  the  concentrations  of  the  salts  used. 
The  ordinate  at  0  represents  the  volume  in  blank  cultures. 


Fig.  5c. — Appearance  of  yeast  cells  in  extreme  concentrations  of  salts. 

Normal  yeast  cells  in  1,  2  and  3,  diseased  yeast  cells  from  extreme  concen- 
trations of  KC1  and  MgCl2  in  4  and  5;  (white)  and  diseased  yeast  cells  from 
extreme  concentrations  of  CaCl2  and  NaCl  in  6,  7  and  8  (black  or  shadowy) 
(X5000). 


1917]        Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast  79 

The  measurements  given  are  the  average  of  five  counts  in  each 
case.  The  volumes  from  which  the  curves  have  been  drawn  in  figure  5 
(a,  b)  have  been  calculated,  for  purposes  of  comparison,  as  though 
the  cells  were  cylindrical. 

Both  from  table  5  (a,  b,  c,  d)  and  the  curves  in  figure  5  (a,  b)  it 
is  evident  that  KC1  and  MgCl2  favor  growth  in  size  up  to  the  most 
favorable  concentration,  beyond  which  the  cells  decrease  in  size  until 
the  extreme  concentration  is  reached,  where  they  dissolve.  Both  NaCl 
and  CaCl2  limit  the  growth  even  in  minute  concentrations,  thus  show- 
ing their  extreme  toxicity  to  yeast  cells. 

Yeast  cells  seem  to  have  remarkable  resistant  power.  Many  of 
them  with  cell  wall  thickened  to  a  heavy  membrane  have  been  found 
in  extreme  concentrations.  Perhaps  this  heavy  membrane  is  formed 
to  resist  the  osmotic  pressure  outside  the  cell.  Besides  some  of  the 
cells  in  these  extreme  concentrations  are  in  norma]  condition  and  are 
even  budding,  thus  showing  the  power  of  adaptability  of  yeast  cells 
to  new  conditions.  After  they  have  become  habituated  to  the  presence 
of  toxic  salts,  they  grow  normally  and  reproduce.  It  is  probably 
owing  to  fhe  adaptability  of  yeast  to  different  conditions  that  the 
same  yeast,  S.  ellipsoideus,  collected  from  various  sources,  shows  dis- 
similar physiological  characters.  Besides  in  many  cases  I  have 
observed  that  the  diseased  yeast  cells  in  extreme  toxicity  of  KC1  and 
MgCl2  form  a  white  membrane  with  normal  cell  contents,  while  those 
of  CaCl2  and  NaCl  form  a  rather  dark  cell  membrane  with  shadowy 
cell  contents.  A  similar  case  to  that  of  Loeb5' 13  may  be  cited  here. 
In  his  experiments  with  sea  urchin  eggs  he  found  two  distinct  phases 
of  cytolysis  which  he  terms  "black  cytolysis"  and  "white  cytolysis." 

With  regard  to  the  effect  of  the  salts  on  the  size  of  the  yeast  cells, 
NaCl  is  the  most  and  KC1  the  least  toxic,  while  CaCl2  and  MgCL 
stand  midway.  The  effect  is  parallel  with  that  of  the  multiplication 
of  cells. 

An  experiment  was  carried  on  with  a  second  culture  of  8.  ellip- 
soideus collected  from  another  source  by  Cruess  and  named  no.  60. 
This  experiment  was  also  made  in  duplicate.  With  this  yeast  potas- 
sium chloride  and  magnesium  chloride  gave  the  same  results  as  with 
no.  66,  but  NaCl  and  CaCl2  showed  a  marked  difference  and  CaCl2  was 
the  most  toxic  of  all.  4M  NaCl  gave  an  appreciable  number  of  yeast 
cells,  while  even  .3M  CaCl2  stopped  the  growth  altogether.  Further, 
the  number  of  yeast  cells  was  much  lower  than  that  of  yeast  no.  66. 
Evidently  yeast  no.  60  was  less  vigorous  than  the  other,  though  other- 
wise there  was  no  fundamental  difference  between  them. 


80  University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 

B.  Experiments  With  Combinations  of  Salts — Antagonistic 

Effects 

The  toxic  effects  of  the  single  salts  KC1,  MgCl2,  CaCL  and  NaCl 
upon  a  wine  yeast,  8.  ellipsoideus,  have  been  shown  in  the  first  part 
of  this  paper.  The  results  of  the  study  indicate  that  the  reactions 
of  yeast  differ  from  those  of  plants,  animals  or  bacteria.  This  second 
part  of  the  paper  gives  the  results  of  an  investigation  to  ascertain  the 
effects  of  various  binary  combinations  of  the  salts  named  upon  the 
same  yeast. 

From  the  four  salts,  six  combinations  of  two  salts  each  are  possible. 
All  of  these  were  tested.  Judging  from  analogous  work  of  other 
investigators  with  animals,  plants  and  bacteria,  it  was  expected  that 
these  salts  would  exhibit  mutually  antagonistic  action,  i.e.,  that  the 
toxicity  of  one  salt  would  be  reduced  by  the  presence  of  another  and 
that  the  total  effect  of  two  salts  together  would  be  less  than  the  sum 
of  their  individual  effects.  In  some  cases  definite  antagonistic  effects 
were  found.  In  others  antagonism  was  not  so  well  defined.  In  a 
few  instances  there  was  no  antagonism  shown. 

In  the  discussion  of  results,  considerable  space  has  been  given  to 
the  findings  of  other  investigators  because  it  was  considered  important 
to  point  out  how  the  effects  on  other  organisms  compare  with  those 
on  yeast.  A  few  words  on  the  development  of  the  idea  of  antagonism 
in  binary  combinations  of  salts  will  be  of  value  as  an  introduction  to 
the  data  in  this  paper. 

Considerable  work  on  the  antagonistic  effects  of  salts  has  been 
done  by  Einger,  Locke,  Howell,  Loeb,  Osterhout,  Overton,  Ostwald, 
Loew,  Lipman  and  others.  That  the  poisonous  effect  of  one  salt  is 
reduced  by  the  addition  of  another  salt  has  been  known  for  a  long 
time,  especially  among  animal  physiologists.  In  this  matter  we  owe 
a  great  deal  of  our  knowledge  to  Loeb,  whose  investigations  brought 
forth  a  large  number  of  unexpected  results.  It  was  he  who  first 
developed  the  theory  that  the  valences  of  metallic  ions  have  consid- 
erable influence  on  their  toxic  and  antagonistic  effects,  and  that  mono- 
valent cations  may  be  antagonized ;by  bivalent,  trivalent  or  tetravalent 
but  not  by  monovalent  cations.  His  results  show  some  parallelism 
to  the  work  of  Linder  and  Picton.*  This  general  statement  does  not 
apply  in  all  cases  to  plants,  animals  and  bacteria,  experimented  upon 
by  various  other  investigators.    Neither  does  it  apply  always  to  yeast. 


*  Hober  and  Gordon,  Beitr.  zur  chem.  physiol.,  vol.  5,  p.  432,  1904,  cited  by 
Osterhout.22 


1917]         Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast  81 

The  experiments  with  binary  salts  were  made  in  the  same  general 
way  as  those  with  simple  salts,  but  with  slight  modifications  of  tech- 
nique. The  flasks  were  arranged  as  before  in  duplicate,  but  in  com- 
bining the  salts  in  different  molecular  concentrations  the  method 
followed  differed  from  those  of  previous  investigators. 

Of  the  two  salts  to  be  tested  for  antagonism,  one  was  weighed  from 
the  minimum  concentration  to  that  of  extreme  toxicity  according  to 
the  molecular  concentration,  and  the  other  was  weighed  and  added 
to  the  former  in  the  reverse  way  in  the  corresponding  flasks.  The 
flasks  containing  the  extreme  concentration  of  each  salt  did  not  receive 
any  addition  of  the  other  salt.  Aside  from  this,  the  methods  of  in- 
oculation, incubation,  and  microscopical  counting  were  the  same  as 
those  described  for  the  single  salts.  Duplicates  were  made  in  all  cases 
and  two  blanks  were  used  in  each  series,  as  checks  on  the  growth  of 
the  yeast  in  the  treated  flasks.  The  same  yeast,  S.  dlipsoideus,  no.  66, 
was  employed  in  these  experiments  as  in  the  ones  with  simple  salts. 
The  results  given  are  therefore  the  average  of  duplicate  experi- 
ments. 


SEEIES  VI— ANTAGONISM  BETWEEN  MAGNESIUM  CHLOKIDE  AND 
CALCIUM  CHLORIDE 

In  this  series  MgCl2  and  CaCl2  were  combined  in  various  mole- 
cular concentrations.  A  series  of  16  Erlenmyer  flasks  was  arranged 
in  duplicate  with  two  blank  cultures.  First,  amounts  of  MgCl2  cor- 
responding to  from  OM  to  2.2M  were  weighed  and  put  in  the  flasks, 
as  was  done  with  the  single  salts.  The  CaCl2  also  was  weighed  ac- 
cording to  its  molecular  concentration  and  put  in  the  same  flasks  in 
reverse  order,  leaving  the  extreme  concentrations  of  each  salt  free 
from  the  addition  of  the  other.  Thus  the  first  two  flasks  received 
.72M  CaCl2  without  any  addition  of  MgCl2 ;  the  second  received  .66M 
CaCl2  and  .001  MgCl2 ;  the  third  .60M  CaCl2  and  .01M  MgCl2,  and  so 
on  to  the  last  couple,  which  contained  only  1.2M  MgCl2  and  no  addition 
of  CaCl2.  The  remaining  flasks  were  combined  in  different  molecular 
concentrations,  as  shown  in  table  1.  Two  blanks  were  taken  to  which 
no  salt  was  added. 

In  order  to  facilitate  the  plotting  of  the  curves,  the  different 
combinations  of  salts  have  been  indicated  by  letters  A,  B,  C,  D,  etc. 
A  represents  the  blank  cultures,  while  the  other  letters  represent  the 
different  molecular  combinations  shown  in  the  table  below: 


82 


University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 


Table  6 — Antagonistic  Effect  Between  MgCl,  and  CaCl0 


No. 

MgClo  vs. 
CaCl2  M.  Cone. 

48  hrs. 

96  hrs. 

144  hrs. 

192  hrs. 

240  hrs. 

A 

.00 

x.0.0 

1,954,000 

6,290,000 

10,556,000 

14,108,000 

16,944,000 

B 

.00 
.00] 

x.72 
x.66 

n 

226,000 
4,520,000 

452,000 

D 

.01 

x.60 

452,000 

2,034.000 

3,842,000 

5,650,000 

E 

.1 

x.48 

8,362,000 

20,860,000 

23,120,000 

24,730,000 

26,842,000 

F 

.2 

x.36 

10,848,000 

26,024,000 

29,706,000 

30,856,000 

31,960,000 

G 

.4 

x.18 

9,718,000 

28,996,000 

32,284,000 

33,974,000 

36,120,000 

H 

.6 

x.06 

8,804,000 

25,286,000 

30,256,000 

31,865,000 

32,556,000 

I 

.8 

x.01 

452,000 

4,972,000 

8,289,000 

10,298,000 

12,684,000 

J 

1.0 

x  .001 

226,000 

1,130,000 

2,260,000 

3,129  000 

T\ 

1.2 

x.00 

A        B 

MgCl2 


D         E         F         G       H 

Concentration  of  salts 


K 
CaCl2 


Fig.  6. — Curves  of  yeast  growth  showing  antagonism  between  MgCL  and 
Cacl2.  The  ordinates  represent  the  number  of  yeast  cells  in  millions  and  the 
abscissae,  the  concentration  of  the  salts  in  combination.  The  ordinates  at  A 
represent  the  number  of  yeast  cells  in  blank  cultures. 


1917]        Ultra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast  Hi', 

From  both  table  6  and  the  curves  in  figure  6  it  is  evident  that 
there  is  a  distinct  antagonism  between  these  two  salts.  For  example, 
in  the  experiments  with  simple  salts  MgCl2  alone  at  .8M  concentration 
allowed  the  growth  of  yeast  cells  up  to  only  8%  millions,  but  in  com- 
bination with  .01M  CaCl2  the  growth  was  increased  up  to  12%  millions, 
i.e.,  50  per  cent  increase.  Similarly,  .6M  CaCL  alone  allowed  an 
increase  to  about  one  millions,  and,  with  the  addition  of  .01  MgCl2, 
an  increase  to  5%  millions,  showing  5%  times  more  growth.  The 
highest  number  in  MgCl2  alone  was  26%  millions  at  .1M,  and  in  CaCl2 
alone  19  millions  at  .01M  concentration.  In  this  binary  combination 
the  highest  number  was  obtained  at  G,  the  point  where  AM.  MgCl2  and 
.18M  CaCL  were  combined  with  a  ratio  of  about  2  :1. 

For  purposes  of  comparison  let  us  now  consider  the  results  obtained 
in  similar  experiments  with  these  four  salts  on  plants,  animals,  and 
bacteria. 

(a)  Plants. — Kearney  and  Cameron8  found  a  distinct  antagonism 
between  Mg  and  Ca  ions  for  higher  plants.  In  their  experiments  with 
leguminous  plants  Lupinus  albus  and  Medicago  sativa  they  found  that, 
for  a  combination  of  these  two  salts,  the  plants  show  about  five  times 
as  much  tolerance  as  for  the  salts  separately.  The  plants  also  dis- 
played a  remarkable  degree  of  tolerance  when  MgS04  was  used  in- 
stead of  MgCL,  thus  showing  in  addition  the  relative  difference  be- 
tween different  anions  of  the  same  salt. 

Loew  and  his  pupils,10' 18  in  their  experiments  with  lower  plants 
(Spirogyra),  have  found  a  strong  antagonism  between  Mg  and  Ca 
ions. 

(6)  Animals. — Loeb2  with  sea  urchins  (blastulae  and  gastrulae) 
found  that  a  mixture  of  MgCl2  (10/8n)  and  CaCL  (10/Sn)  will  allow 
them  to  swim  for  about  forty-eight  hours,  while  each  of  the  salts 
singly  at  the  same  concentration  is  extremely  poisonous  and  kills  the 
animals.  The  same  investigator15  working  with  a  jellyfish  (Poly or- 
chis) has  shown  that  the  addition  of  a  small  quantity  of  CaCL  to  a 
mixture  of  NaCl  and  MgCl2  favors  the  normal,  rhythmical  contrac- 
tions, while  MgCL  alone  stops  them  altogether.  Contrary  to  the 
above  results,  Loeb12  in  his  experiments  with  frogs  has  found  that  a 
combination  of  Mg  and  Ca  ions  completely  inhibits  the  rhythmical 
muscular  contractions.  This  has  been  corroborated  by  Anne  Moore,7 
in  her  experiments  with  the  contraction  of  the  lymph  hearts  of 
frogs. 

Lillie6   has   found   that   the   ciliary  movement   of    the   larvae   of 


84  University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 

Arenicola  goes  on  normally  for  a  time  in  a  mixture  of  MgCl2  and 
CaCl2  with  the  ratio  of  4:1  at  10/8n  concentration,  though  either  of 
the  two  salts  used  alone  would  stop  it  entirely.  Matthews,11  in  his 
work  with  the  development  of  embryos  in  the  eggs  of  Fundulus,  found 
a  distinct  antagonism  between  Mg  and  Ca. 

Meltzer  and  Auer21  have  shown  with  rabbits  and  a  monkey  that 
the  poisonous  action  of  MgCl2  in  subcutaneous  injection  is  similarly 
diminished  by  the  injection  of  CaCl2.  They  found  also  a  strong 
antagonism  between  the  nitrates,  acetates  and  sulfates  of  these  two 
salts  respectively. 

(c)  Bacteria. — Lipman,23' 24  with  a  soil  bacterium,  Bacillus  siib- 
tilis,  found  little  or  no  antagonism  between  the  two  salts,  but,  on  the 
contrary,  the  addition  of  one  salt  to  the  other  was  found  to  be  more 
toxic  than  either  of  the  two  salts  used  alone. 

All  of  the  above  mentioned  experiments,  except  those  of  the  three 
cases  of  Lipman,  Loeb,  and  Anne  Moore,  are  in  agreement  with  the 
antagonistic  effects  between  Mg  and  Ca  ions  that  occur  with  yeast. 
In  addition,  it  may  be  noted  here  that  the  antagonistic  effect  between 
MgCl2  and  CaCl2  with  yeast  has  been  found  to  be  the  strongest  of  all 
the  combinations.  This  corroborates  the  opinion  advanced  by  Loew 
that  there  is  a  strong  antagonism  between  calcium  and  magnesium 
both  with  plants  and  animals.10 


SERIES  VII— ANTAGONISM  BETWEEN  POTASSIUM  CHLORIDE  AND 
CALCIUM  CHLORIDE 

In  this  series  the  experiments  were  carried  on  in  the  same  way  as 
with  MgCl2  and  CaCl2.  Table  7  and  the  curves  in  figure  7  show 
there  is  a  distinct  antagonism  between  the  two  salts.  In  this  case 
marked  antagonism  was  found  on  the  side  of  CaCl2,  but  little  or  none 
on  the  side  of  KC1.  For  example,  the  combination  of  .001M  KC1  with 
.66M  CaCl2  allowed  the  yeast  to  grow  up  to  6%  millions,  while  in 
CaCl2  at  .6  alone  the  yeast  was  found  to  increase  only  up  to  about  one 
million.  Thus  there  was  6!/2  times  as  much  growth  where  the  KC1 
was  present.  But,  on  the  other  hand,  the  combination  of  .001M  CaCl2 
to  2.0M  KC1  did  not  accelerate  the  growth.  This  unexpected  result 
may  be  accounted  for  by  the  fact  that  the  higher  concentrations  of 
KC1  being  very  high  in  comparison  to  the  small  concentrations  of 
CaCl2  the  latter  was  not  sufficient  to  reduce  the  toxicity  of  the  KC1 
at  such  a  high  concentration.    It  is  also  very  probable  that  a  concen- 


1917]        Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast  85 

tration  of  2.0M  KC1  exerts  a  strong  osmotic  effect  and  that  the  toxicity 
is  due  to  osmotic  influences  rather  than  to  the  usual  toxicity  of  the 
ion  itself.  If  this  were  true  we  would  expect  little  antagonism  from 
other  salts. 

Loeb12  in  his  experiments  with  a  jellyfish  (Gonionemus)  met  with 
a  similar  difficulty.  In  this  case  the  KC1  concentration  was  so  high 
that  a  small  concentration  of  NaCl  did  not  remove  the  toxicity,  and 
so  the  combination  inhibited  the  contraction  of  the  animal,  while  the 
same  concentrations  used  in  the  case  of  another  kind  of  fish,  Fundulus, 
allowed  the  development  of  embryos  in  the  eggs.  He  has  pointed  out 
the  fact  that  in  the  embryos  of  Fundulus  the  solutions  in  which  cleav- 
age proceeds  normally  interferes  seriously  with  the  heartbeat  of  Coni- 
onemus,  if  the  proportion  of  KC1  exceeds  a  certain  limit.  In  this 
instance  we  find  proof  of  the  fact  that  in  the  same  organism  cell- 
division  and  muscular  contractility  are  influenced  by  entirely  different 
combinations  of  ions,  and  therefore  these  vital  activities  must  depend 
on  widely  different  chemical  constitutions.  However,  the  highest 
growth  in  the  case  of  yeast  was  obtained  at  H,  where  .6M  KC1  and 
.36M  CaCl2  have  been  combined,  a  ratio  of  about  2:1.  In  the  case  of 
KCl  alone  the  highest  growth  was  obtained  at  .2M  concentration, 
allowing  growth  up  to  3OV2  millions  per  c.c.  CaCl2  allowed  growth 
up  to  19  millions  at  .01M  concentration. 

Table  7 — Antagonistic  Effects  Between  KCl  and  CaCl, 


No. 

KCl  vs. 
CaCl2  M.  Cone. 

48  hrs. 

96  hrs. 

144  hrs. 

192  hrs. 

240  hrs. 

A 

.00 

x.00 

2,101,000 

8,589,000 

13,908,000 

16,896,000 

17,520,000 

B 

.00 
.00] 

x.72 
x.66 

<^ 

226,000 

2,034,000 

3,985,000 

6,722,000 

D 

.01 

x.60 

452,000 

4,972,000 

13,315,000 

]  8,604,000 

22,720,000 

E 

.1 

x.54 

4,020,000 

10,328,000 

20,245,000 

23,266,000 

25,120,000 

F 

.2 

x.48 

5,558,000 

12,840,000 

22,190,000 

26,880,000 

29,380,000 

G 

.4 

x.42 

3,034,000 

12,840,000 

26,852,000 

19,126,000 

32,285,000 

H 

.6 

x.36 

1,017,000 

8,398,000 

13,645,000 

28,904,000 

34,500,000 

I 

.8 

x.30 

226,000 

4,256,000 

10,250,000 

14,966,000 

18,732,000 

,T 

1.0 

x.24 

2,965,000 

6,126,000 
3,986,000 

9,551,000 

16,159,000 

K 

1.2 

x.18 

1,130,000 

5,410,000 

7,119,000 

T, 

1.4 

x.12 

452,000 

2,550,000 

2,712,000 

4,438,000 

M 

1.6 

x.06 

904,000 

1,130,000 

3,906,000 

TsJ 

1.8 
2.0 

x.01 
x.001 

452,000 

904,000 
226,000 

2,652,000 

O 

1,130,000 

P 

2.2 

x.00 

86 


University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 


C         D 
KC1 


F         G        H         I  J         K 

Concentration  of  salts 


M         N         O 

CaClo 


Fig.  7. — Curves  of  yeast  growth  showing  antagonism  between  KC1  and  CaCL. 
The  ordinates  represent  the  number  of  yeast  cells  in  millions  and  the-  abscissae, 
the  concentration  of  salts  in  combination.  The  ordinate  at  A  represents  the 
number  of  yeast  cells  in  blank  cultures. 

For  comparison  with  these  results,  a  number  of  cases  dealing  with 
plants,  animals  and  bacteria  may  be  cited  below : 

(a)  Plants. — Osterhout22  has  shown  that  for  higher  plants  a  com- 
bination of  100  c.c.  KC1  and  5  c.c.  CaCL  at  the  concentration  of  .12M 
is  best  suited  for  the  highest  development  of  roots.  Benecke19  has 
shown  that  for  lower  plants  (Spirogyra)  the  harmful  effect  of  the  K 
ion  is  very  distinctly  antagonized  by  the  addition  of  the  Ca  ion  at  a 
certain  definite  concentration. 


1917]        Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast  87 

(b)  Animals. — In  regard  to  the  development  of  embryos  in  the 
eggs  of  Fundiilus,  Loeb1  has  met  with  a  marked  antagonism  between 
the  two  salts,  using  75  c.c.  of  KC1  (5/8n)  and  25  c.c.  of  CaCl2  (10/8n). 
This  combination  allowed  the  development  of  a  number  of  embryos, 
while  in  the  same  concentration  of  KC1  alone  no  development  was 
shown.  He  also  obtained  a  similar  result  with  the  muscular  contrac- 
tion of  a  jellyfish  (Poly orchis),15  thus  showing  an  antagonistic  effect 
between  the  two  salts.  The  same  investigator3  in  his  experiments  with 
the  hydromedusa  Gonionemus  has  shown  that  the  combination  of  K 
ion  (5/8n)  and  Ca  ion  (10/8n)  is  poisonous  to  the  animals.  Anne 
Moore7  obtained  a  similar  result  in  her  experiments  on  the  contraction 
of  the  lymph  heart  of  frogs. 

Meltzer  and  Auer21  have  shown  that  with  rabbits  and  a  monkey 
in  subcutaneous  injection  there  is  a  limited  antagonism  between  the 
two  salts.  Matthews11  with  Fundulus  met  with  a  similar  result.  He 
found  that  at  the  dilution  of  M/1600  CaCL  to  6/8n  KC1  the  develop- 
ment of  embryos  in  the  eggs  was  found  to  be  the  best. 

Lillie6  found  that  with  the  larvae  of  Arenicola  the  ciliary  activity 
went  on  when  he  used  97.5  c.c.  CaCL  (10/8n)  and  2.5  c.c.  KC1  (5/8n), 
showing  an  antagonism  between  the  two  salts. 

(c)  Bacteria. — Lipman23  has  shown  that  for  Bacillus  subtilis  the 
highest  production  of  ammonia  is  found  at  the  point  where  100  c.c. 
KC1  and  5  c.c.  CaCL  at  the  concentration  of  .35M  is  used,  thus  showing 
a  distinct  antagonism  between  the  two  salts.  His  work  has  a  striking 
similarity  to  that  of  Osterhout  on  wheat. 

Summarizing  the  antagonism  between  K  and  Ca,  it  may  be  said 
that  the  toxicity  of  high  concentrations  of  Ca  is  greatly  reduced  by 
the  presence  of  K,  but  that  the  toxicity  of  high  concentrations  of  K 
is  not  appreciably  reduced  by  small  amounts  of  Ca.  The  optimum 
ratio  of  KC1  to  CaCL  was  about  2 :1  for  yeast. 


SERIES  VIII— ANTAGONISM  BETWEEN  MAGNESIUM  CHLOEIDE  AND 

SODIUM  CHLOEIDE 

The  experiments  in  this  series  were  carried  on  in  the  same  way  as 
the  others.  Both  table  8  and  the  curves  in  figure  8  show  that  there 
is  a  distinct  antagonism  between  these  two  salts.  The  highest  growth 
in  this  case  was  found  at  G,  the  point  where  .4M  MgCL  and  .06M 
NaCl  were  combined,  a  ratio  of  about  6  :1.  As  already  shown,  when 
used  singly  MgCL  allows  the  highest  growth  at  .1M,  i.e.,  26y2  millions, 


88 


University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 


Table  8 — Antagonistic  Effects  Between  MgClo  and  NaCl 


No. 

A 

B 

C 

D 

E 

F 

G 

H 

I 

J 

K 


MgClo  vs. 
NaCl    M.  Cone 

.00     X.00 

.00    x.208 

.001  x  .180 


.01 

.1 

.2 

.4 

.6 

.8 
1.0 
1.2 


x.160 

x.128 

x.096 

x.064 

x.032 

x.01 

x.001 

x.00 


48  hrs. 
3,100,000 


226,000 
3,250,000 
5,424,000 
2,260,000 
1,582,000 

904,000 


96  hrs. 
7,644,000 


452,000 

6,212,000 

10,396,000 

12,656,000 

8,684,000 

3,102,000 

904,000 


144  hrs. 
13,686,000 


452,000 
11,201,000 
16,368,000 
20,696,000 
14,956,000 
6,358,000 
1,872,000 


192  hrs. 
15,203,000 

226,000 
678,000 
14,258,000 
21,690,000 
25,882,000 
17,009,000 
10,605,000 
3,896,000 


240  hrs. 
17,009,000 

226,000 
1,130,000 
17,255,000 
25,793,000 
28,890,000 
21,583,000 
15,430,000 
4,276,000 


A 
MgCl2 


D    ■      E  F         G        H 

Concentration  of  salts 


NaCl 


Fig.  8. — Curves  of  yeast  growth  showing  antagonism  between  MgCL  and 
NaCl.  The  ordinates  represent  the  number  of  yeast  cells  in  millions  and  the 
abscissae,  the  concentration  of  salts  in  combination.  The  ordinate  at  A  repre- 
sents the  number  of  yeast  cells  in  blank  cultures. 


1917]        Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast  89 

and  NaCl  at  .001M,  i.e.,  18  millions.  But  in  combination  the  two  salts 
permit  the  highest  growth  of  29  millions  per  c.c.  at  .4M  and  .06M 
respectively. 

The  antagonism  between  these  two  salts  in  the  case  of  yeast  is 
found  very  distinctly  at  both  ends  of  the  curves.  For  example,  .1M 
NaCl  alone  shows  a  growth  of  scarcely  more  than  one  million,  while 
in  combination  with  .1M  MgCl2  it  shows  over  17  millions,  or  17  times 
as  much.  On  the  other  hand,  .8M  MgCl2  alone  allowed  a  growth 
of  about  8y2  millions,  while  in  combination  with  .01M  NaCl  the 
growth  was  increased  to  about  15%  millions,  or  about  twice  as 
much. 

In  comparison  with  these  results,  a  number  of  cases  dealing  with 
the  effects  of  combinations  of  MgCl2  and  NaCl  on  plants,  animals  and 
bacteria  are  cited  below. 

(a)  Plants. — Osterhout5  found  a  distinct  antagonism  between  the 
two  salts  with  the  growth  of  a  fungus  (Botrytis  cinerea).  He  found 
that  15.M  NaCl  alone  was  very  toxic,  but  that  when  this  concentration 
of  NaCl  was  combined  with  .4  M  MgCl2  the  toxicity  was  much  re- 
duced. He  also  found  with  wheat  that  neither  NaCl  nor  MgCl2  at 
.12M  alone  allowed  root  development,  but  in  a  combination  in  the 
proportion  of  100  c.c.  NaCl  to  75  c.c.  MgCl2  the  root  developed  very 
well.  The  same  investigator  obtained  a  negative  result  with  green 
algae.20 

Kearney  and  Cameron8  with  Lupinus  albus  and  Medicago  sativa 
have  shown  that  the  addition  of  MgCl2  to  NaCl  raised  the  tolerance 
of  these  plants  to  the  latter  3-10  times. 

(b)  Animals. — Loeb12  with  Fundulus  has  found  that  in  a  mixture 
of  98  c.c.  5/8n  NaCl  and  2  c.c.  10/8n  MgCl2  all  the  eggs  develop 
embryos,  while  the  same  salts  alone  at  the  same  concentration  are 
extremely  toxic.  Even  an  equal  proportion  of  the  two  salts  in  the 
same  concentration  allowed  about  75  per  cent  of  the  embryos  to  de- 
velop. He  also  found  a  similar  antagonism  with  a  sea  urchin  (Ar- 
bacia)  and  a  jellyfish  {Poly orchis). 

Lillie6  found  that  with  the  larvae  of  Arenicola  the  ciliary  move- 
ment continued  for  a  time  when  he  added  10  c.c.  MgCl2  (10/8n)  to 
90  c.c.  NaCl  (5/8n),  while  the  same  concentrations  of  NaCl  alone 
would  stop  it  immediately.  Matthews  with  Fundulus  found  an  an- 
tagonism between  the  two  salts. 

Ostwald,13  however,  with  fresh-water  Grammarus  obtained  con- 
trary results.    In  this  case  a  combination  of  the  two  salts  was  found 


90  University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 

to  be  more  toxic  than  NaCl  alone,  isotonic  with  sea  water  (2.7  per 
cent  NaCl  in  sea  water  or  about  .4M  NaCl). 

(c)  Bacteria. — Lipman23  with  Bacillus  subtilis  obtained  a  result 
similar  to  that  of  Osterhout.  A  mixture  of  the  same  concentration 
of  MgCl2  and  NaCl  (.35M)  in  the  ratios  of  10:1  gave  the  maximum 
production  of  ammonia. 

To  summarize  the  results  of  these  experiments,  it  may  be  said 
that  there  is  a  distinct  antagonism  between  MgCl2  and  NaCl,  which 
is  evident  on  both  ends  of  the  curves  in  figure  8.  In  this  case  the 
yeast  agrees  with  the  observations  on  plants,  animals  and  bacteria 
except  in  the  two  instances  cited  above  in  regard  to  fresh-water 
Grammar  us  and  green  algae. 


SERIES  IX— ANTAGONISM  BETWEEN  POTASSIUM  CHLORIDE  AND 

SODIUM  CHLORIDE 

In  this  series  the  flasks  were  arranged  as  before.  It  has  been 
pointed  out  by  Loeb  that  two  salts  with  ions  of  like  valence,  especially 
in  the  case  of  monovalent  ions,  do  not  antagonize  the  toxicity  of  each 
other,  but  rather  show  a  moderately  increased  toxicity  when  com- 
bined. This  is  evident  with  yeast,  as  is  shown  by  table  9  and  the 
curves  in  figure  9.  The  highest  growth  in  this  case  was  found  at  F, 
where  .2M  KC1  and  .12M  NaCl  have  been  combined,  having  a  ratio 
of  about  2 :1.  KC1  alone  at  .2M  concentration  allows  the  growth 
about  iy2  times  that  found  in  this  combination.  Thus  the  antag- 
onism of  NaCl  for  KC1  is  found  to  be  negative.  But,  on  the  other 
hand,  there  is  a  distinct  antagonism  of  KC1  for  NaCl.  For  example, 
NaCl  alone  at  .17M  concentration  hardly  allowed  any  growth,  but  in 
combination  with  .01M  KC1  the  growth  was  accelerated  up  to  about 
15  millions,  thus  showing  a  distinct  antagonism.  The  reason  of  this 
unexpected  negative  result  on  the  side  of  KC1  is  perhaps  the  same 
that  I  have  suggested  in  the  case  of  KC1  and  CaCh  in  Series  II. 

For  comparison  with  these  results,  a  number  of  cases  dealing  with 
plants,  animals  and  bacteria  are  cited  below: 

(a)  Plants. — Osterhout22  with  wheat  (Early  Genesee)  has  found 
a  slight  antagonism  between  K  and  Na  ions.  But  in  his  work17  on 
a  green  alga  he  obtained  a  negative  result  using  3/8M  concentration 
of  two  salts  in  combination. 

(~b)  Animals. — Loeb1  with  Fundulus  found  a  slight  antagonism 
between  the  K  and  the  Na  ion  in  relation  to  the  development  of  em- 


1917]        Alitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast 


91 


Table  9 — Antagonistic  Effects  Between  KCl  and  NaCl 


No. 

KCl  vs. 
NaCl    M.  Cone 

48  hrs. 

96  hrs. 

144  hrs. 

192  hrs. 

240  hrs. 

A 
B 

.00 
.00 
.00] 
.01 

x.00 

x.208 

L  x  .192 

x.176 

1,954,000 

6,290,000 

10,556,000 

14,108,000 

16,944,000 

f! 

226,000 
7,184,000 

1,808,000 
9,256,000 

6,780,000 
12,176,000 

7,888,000 
14,952,000 

D 

2,678,000 

E 

.1 

x.160 

5,424,000 

10,786,000 

14,690,000 

16,922,000 

18,566,000 

F 
G 
H 

#2 
.4 
.6 

x.144 
x.128 
x.112 

2,938,000 

2,260,000 

678,000 

12,339,000 
7,838,000 
6,780,000 

15,942,000 
11,526,000 

8,678,000 

18,206,000 
15,830,000 
12,687,000 

21,250,000 
17,248,000 
13,266,000 

I 
J 

.8 
1.0 

x.096 
x.080 

226,000 

5,650,000 
4,156,000 
2,906,000 

7,205,000 
5,882,000 

10,256,000 
8,120,000 
7,750,000 

12,984,000 
9,886,000 
8,^05,000 

K 

1.2 

x.064 

4,900,000 

Ti 

1.4 

x.048 

1,130,000 

3,390,000 

1,130,000 

226,000 

4  968  000 

6  983  000 

M 

1.6 
1.8 

x.032 
x.010 

452,000 

2,260,000 
1,130  000 

4,452,000 

2,960,000 

904  000 

N 

O 

2.0 

2.2 

x  .001 
x.00 

452,000 

P 

a  s 


B         C 
KCl 


F         G        H         I  J 

Concentration  of  salts 


M         N         O 
NaCl 


Fig.  9. — Curves  of  yeast  growth  showing  antagonism  between  KCl  and 
Nacl.  The  ordinates  represent  the  number  of  yeast  cells  in  millions  and  the 
abscissae,  the  concentration  of  salts  in  combination.  The  ordinate  at  A  repre- 
sents the  number  of  cells  in  blank  cultures. 


92  University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 

bryos  in  the  eggs.  He  also  found  a  similar  result  with  sea-urchins, 
Hydromedusa  gonionemus,  and  a  jellyfish,  Polyorchis. 

Lillie6  found  that  with  the  larvae  of  Arenicola  the  ciliary  move- 
ment goes  on  in  a  solution  containing  20  parts  of  NaCl  (5/8n)  and 
8  parts  of  KC1  (5/8n),  while  each  salt  used  alone  stops  the  movement 
altogether. 

Ostwald13  with  fresh-water  Gammarus  has  shown  that  there  is  a 
distinct  antagonism  between  K  and  Na  ions  in  regard  to  the  duration 
of  life  of  that  animal.  Matthews11  has  found  that  it  takes  twice  as 
much  KC1  to  neutralize  the  toxicity  of  NaCl  in  the  case  of  the  devel- 
opment of  embryos  in  the  eggs  of  Fundulus.  This  is  rather  similar 
to  the  case  of  yeast,  where  it  takes  .2M  KC1  to  neutralize  the  toxicity 
of  .14M  NaCl  to  allow  the  highest  growth. 

(c)  Bacteria. — Lipman23  with  Bacillus  subtilis  has  found  that  none 
of  the  combinations  of  these  two  salts  gives  as  favorable  conditions 
for  growth  as  is  found  with  each  salt  alone  at  the  same  concentration, 
thus  showing  non-antagonism  between  the  two  salts. 

To  summarize  the  results  in  this  experiment,  it  may  be  said  that 
with  yeast,  like  valences  prevent  the  antagonistic  effects,  contrary  to 
what  was  found  by  Lipman  with  soil  bacteria,  but  in  accordance  with 
the  results  of  Osterhout  with  wheat,  Loeb  with  Fundulus,  and  other 
investigators  with  other  organisms.  The  yeast  agrees  in  this  case  with 
all  the  above-mentioned  cases  except  with  that  of  green  algae  tested 
by  Osterhout  and  that  of  Bacillus  subtilis  by  Lipman. 


SERIES  X— ANTAGONISM  BETWEEN  POTASSIUM  CHLORIDE  AND 
MAGNESIUM  CHLORIDE 

The  experiments  in  this  series  were  conducted  like  the  others.  The 
highest  growth  in  this  case  was  found  at  H,  the  point  where  .6M  KC1 
and  .5M  MgCl2  were  combined  in  a  ratio  of  about  1:1.  In  the  case  of 
simple  salts  KC1  alone  at  .2M  concentration  allowed  the  highest  growth 
up  to  about  30i/2  millions  and  MgCl2  at  .1M  about  26y2  millions.  KC1 
alone  at  .6M  and  MgCL  at  .5M  permitted  the  growth  of  yeast  more 
than  is  found  in  this  combination  at  H.  But  this  indicates  a  mild 
antagonism,  because  the  toxic  effect  was  less  than  the  sum  of  the 
separate  toxic  effects  of  the  two  salts  used  alone.  Distinct  antag- 
onism to  the  effects  of  MgCl2  is  shown  by  KC1,  but  not  the  converse. 
For  example,  .8M  MgCl2  alone  allows  the  yeast  to  grow  only  to 
8  millions,  while  in  the  combination  with  .1M  KC1  the  growth  has 


1917]        Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast 


93 


been  increased  to  HV2  millions.  On  the  other  hand,  the  smaller  con- 
centrations of  MgClo  with  higher  concentrations  of  KC1  did  not  show 
any  antagonism.  The  reason  for  this  unexpected  result  is  perhaps 
that  previously  mentioned  in  the  case  of  KC1  vs.  CaCL,  in  Series  VII. 


Table  10 — Antagonistic  Effects  Between  KCl  and  MgCl., 


KCl  vs. 

No. 

MgCl2 

M. 

Cone. 

48  hrs. 

96  hrs. 

144  hrs. 

192  hrs. 

240  hrs. 

A 

.00 

X 

.00 

2,356,000 

9,701,000 

12,170,000 

14,890,000 

17,526,000 

B 

.00 
.00] 

X 

y 

1.2 
1.0 

n 

226,000 

1,130,000 

2,260,000 

3,845,000 
11,560,000 

D 

.01 

X 

.9 

226,000 

1,356,000 

6,780,000 

10,070,000 

E 

.1 

X 

.8 

1,130,000 

6,780,000 

7,408,000 

10,975,000 

12,180,000 

F 

.2 

X 

.7 

1,356,000 

7,458,000 

11,578,000 

13,449,000 

14,328,000 

G 

.4 

X 

.6 

2,486,000 

4,838,000 

7,006,000 

14,690,000 

15,500,000 

H 

.6 

X 

.5 

904,000 

3,816,000 

5,296,000 

12,850,000 

16,280,000 

I 

.8 

X 

.4 

678,000 

2,612,000 

4,852,000 

8,286,000 

9,856,000 

J 

1.0 

X 

.3 

452,000 

2,260,000 

3,706,000 

5,463,000 

5,902,000 

K 

1.2 

X 

.2 

452,000 

2,040,000 

3,295,000 

4,895,000 

5,240,000 

L 

1.4 

X 

.1 

678,000 

1,926,000 

2,940,000 

3,656,000 

4,864,000 

M 

1.6 

X 

.05 

226,000 

904,000 

3,050,000 

3,006,000 

4,628,000 

N 

1.8 

X 

01 

226,000 

2,260,000 

2,990,000 

3,862,000 

O 

2.0 
2.2 

X 

X 

.001 
.00 

2,226,000 

1,130,000 

P 

B         C 
KCl 


F         G        H         I  J 

Concentration  of  salts 


M         N 
MgCl2 


Fig.  10. — Curves  of  yeast  growth  showing  antagonism  between  KCl  and 
MgCl2.  The  ordinates  represent  the  number  of  yeast  cells  in  millions  and  the 
abscissae,  the  concentration  of  salts  in  combination.  The  ordinate  at  A  repre- 
sents the  number  of  cells  in  blank  cultures. 


94  University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 

For  comparison  with  these  results  a  few  cases  may  be  cited  as 
follows : 

(a)  Plants. — Osterhout20  with  wheat  (Early  Genesee)  has  shown 
that  the  root  develops  better  in  a  solution  having  100  c.c.  KC1  and 
7.5  c.c.  MgCL  at  .12M  concentration  than  in  KC1  alone.  He  also 
found  with  a  marine  alga,20  Enteromorpha  hopkirkii,  that  both  salts 
are  poisonous  when  used  alone,  but  a  combination  in  the  proportion 
of  100  c.c.  MgCL  and  40  c.c.  KC1  allows  considerable  growth.  He 
found  a  similar  antagonism  with  liverworts.20 

(b)  Animals. — Matthews11  found  with  Fundulus  that  in  order  to 
permit  development  of  the  embryos  in  the  eggs  at  the  concentration 
of  33/48n  KC1  at  least  about  M/160  MgCL  is  needed.  He  also  found 
that  a  solution  of  6/8n  KC1  requires  M/80  MgCL  to  give  the  best 
result. 

(c)  Bacteria. — Lillie6  has  shown  that  a  combination  of  10/8n 
MgCL  and  5/8n  KC1  allows  the  ciliary  activity  of  the  larvae  of 
Arenicola,  which  is  stopped  when  one  salt  is  used  alone. 

To  summarize,  it  may  be  said  that  a  distinct  antagonism  was  found 
by  Osterhout  with  higher  and  lower  plants  and  by  Matthews  and 
Lillie  with  animals.  With  yeast  a  slight  antagonism  is  found,  which 
is  shown  on  the  curves  in  figure  10  on  the  side  of  MgCL. 


SERIES  XI— ANTAGONISM  BETWEEN  CALCIUM  CHLORIDE   AND 
SODIUM  CHLORIDE 

The  plan  of  this  series  of  experiments  was  the  same  of  that  of  the 
others.  In  the  case  of  simple  salts  both  CaCL  and  NaCl  were  found 
to  be  very  toxic,  and  it  may  be  owing  to  this  extreme  toxicity  that 
the  combinations  of  the  two  salts  showed  increased  toxicity.  Both 
from  table  11  and  the  curves  in  figure  11  it  is  evident  that  this  toxicity 
is  very  marked.  The  highest  growth  was  found  at  E,  where  .1M  CaCL 
and  .12M  NaCl  have  been  combined  in  the  ratio  of  1:1.  But  even 
here  the  number  of  yeast  cells  went  up  only  to  8  millions,  which  is 
far  below  the  highest  growth  obtained  when  the  salts  were  used  alone. 
However,  CaCL  shows  slight  antagonism  to  the  toxicity  of  NaCl,  for 
example,  .1M  NaCL  alone  allows  the  growth  only  to  one  million, 
while  in  combination  with  .1M  CaCl  it  reached  more  than  8  millions. 
On  the  whole,  however,  both  from  the  table  and  the  curves  it  is  evident 
that  the  combinations  of  the  two  salts  are  more  toxic  than  the  single 
salts. 


1917]         Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast 


95 


Table  11 — Antagonistic  Effects  Between  CaCl2  and  NaCl 


No. 
A 
B 
C 
D 
E 
F 
G 
H 
I 
J 
K 


CaCl2  vs. 
NaCl    M.  Cone 

.00    x.00 

.00    x.208 

.001  x. 18 


.01 

.1 

.2 

.3 

.4 

.5 

.6 

.7 


x.16 

x.12 

x.09 

x.06 

x.03 

x.01 

x.001 

x.00 


48  hrs. 

2,356,000 

226,000 
226,000 
226,000 


96  hrs. 
9,381,000 

226,000 
1,582,000 
1,808,000 
1,356,000 

226,000 


144  hrs. 
12,172,000 

226,000 
3,390,000 
5,650,000 
3,482,000 
1,130,000 
.  226,000 


192  hrs. 

14,890,000 

904,000 
4,682,000 
7,910,000 
4,520,000 
5,650,000 
3,390,000 


240  hrs. 
17,108,000 

1,130,000 
5,842,000 
8,290,000 
7,042,000 
6,820,000 
4,526,000 
452,000 


A   B 
CaCl2 


D    E    F    G   H 

Concentration  of  salts 


J         K 

NaCl 


Fig.  11. — Curves  of  yeast  growth  showing  effects  of  NaCl  on  CaCL.  The 
ordinates  represent  the  number  of  yeast  cells  in  millions  and  the  abscissae,  the 
concentration  of  salts  in  combination.  The  ordinate  at  A  represents  the 
number  of  yeast  cells  in  blank  cultures. 


For  comparison  with  other  organisms  the  following  cases  are  cited : 
(a)  Plants. — Osterhout5  with  wheat  found  a  distinct  antagonism 
between  the  two  salts.  He  obtained  a  similar  result  with  green  algae 
in  which  he  used  100  c.c.  NaCl  and  10  c.c.  CaCL  at  the  concentration 
of  3/8M.  Kearney  and  Cameron,8  with  leguminous  plants,  found  that 
a  combination  of  the  two  salts  increased  the  tolerance  of  the  plants  for 
CaCl2  three  times. 


96  University  of  Calif ornia  Publications  in  Agricultural  Sciences         [Vol.  3 

(b)  Animals. — Loeb1  with  Hydromedusa  Gonionemus  has  shown 
that  a  combination  of  10/8n  CaCl2  and  5/8n  NaCl  is  harmless  to 
animals.  He15  also  found  a  distinct  antagonism  with  a  jellyfish, 
Polyorchis,  using  50  c.c.  NaCl  and  1  c.c.  CaCl2,  which  allowed  the 
animal  to  swim,  while  NaCl  alone  was  poisonous.  The  same  investi- 
gator found  a  distinct  antagonism  between  these  two  salts  working 
with  the  development  of  embryos  in  the  eggs  of  the  Fundulus.  Anne 
Moore7  with  the  contraction  of  the  lymph  heart  of  frogs  and  Lingle4 
with  that  of  the  turtle's  heart  noted  similar  phenomena,  thus  corrob- 
orating the  work  of  Loeb. 

Lillie6  working  with  the  larvae  of  Arenicola  has  found  a  distinct 
antagonism  between  Ca  and  Na  ions.  MacCallum14  found  the  same 
with  his  experiments  on  cathartics. 

Meltzer  and  Auer21  found  a  distinctly  antagonistic  effect  with 
animals  in  subcutaneous  injections.  Ostwald13  wth  fresh-water  Gram- 
marus  found  a  strong  antagonism  between  NaCl  and  CaCL  in  regard 
to  the  duration  of  life  of  that  animal.  Finally,  Matthews11  has  shown 
that  there  is  a  slight  antagonism  between  the  two  salts  in  the  develop- 
ment of  embryos  in  the  eggs  of  Fundulus. 

(c)  Bacteria. — Lipman24  with  Bacillus  subtilis  found  a  marked 
lack  of  antagonism  between  the  two  salts.  In  his  case  any  combi- 
nation of  the  two  salts  at  .35M  concentration  was  found  to  be  more 
poisonous  than  a  single  salt. 

All  these  experiments  except  that  of  Lipman  show  that  there  is 
antagonism  between  CaCl2  and  NaCl.  The  yeast  agrees  very  mark- 
edly with  Bacillus  subtilis  in  showing  little  or  no  antagonism  between 
the  two  salts,  CaCl2  and  NaCl2. 


1917]         Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast  97 


Relative  Antagonisms  of  Various  Combinations 

Table  12  is  intended  to  show  the  relative  antagonisms  of  the 
various  combinations.  The  data  used  in  constructing  the  table  are 
the  final  counts  in  each  flask. 

The  average  of  the  counts  in  all  the  check  flasks  is  taken  as  the 
basis  from  which  to  estimate  the  influence  of  the  various  salts  and  of 
their  combinations.    The  calculation  is  made  as  follows : 

Yeast  growth  in  check  flasks  =  17  (millions). 

Yeast  growth  with  single  salt  no.  1  =  a. 

Yeast  growth  with  single  salt  no.  2  =  o. 

Yeast  growth  with  combination  no.  1  +  2  =  c. 

Toxicity  —  expected  =  (17  —  a)  +  (17  —  &). 

Toxicity  —  observed  =  17  —  c. 

Antagonism  of  combinations*  ■=  (17  —  a)  +  (17  —  o) — .(17  —  c). 

m ' .  Antagonism  ==  17  +  c  —  a  —  o. 


Table  12 — Range  of  Antagonism  of  the  Binary  Combinations  Calculated 
From  the  Last  Microscopical  Count* 

No.   MgCl2  X  CaCl2     KC1  X  CaCl2    MgCl2  X  NaCl    KC1  X  NaCl      KOI  X  MgCl2     CaCl2  X  NaCl 
At    17,000,000       17,000,000       17,000,000       17,000,000       17,000,000     17,000,000 

B       

C       4,000,000       5,000,000         4,000,000       

D    4,000,000   17,000,000   8,000,000   10,000,000   4,000,000 

E  27,000,000   17,000,000    8,000,000    9,000,000    9,000,000   7,000,000 

F  24,000,000   18,000,000   25,000,000    8,000,000    7,000,000   7,000,000 

G  20,000,000   30,000,000   27,000,000    7,000,000   16,000,000   6,000,000 

H   21,000,000   31,000,000   14,000,000    7,000,000    9,000,000   

I    7,000,000    8,000,000   11,000,000   10,000,000   

J    1,000,000    1,000,000    2,000,000   10,000,000   

K 1,000,000   

L   

M 

N   

O 

P   


f  Millions  on  average. 

*  These  results  are  shown   graphically  by  the   curves  in   figure   12. 

*  This  defines  ' antagonism'  as  the  difference  between  the  expected  and  the 
observed  toxicity. 


The  curves  have  been  drawn  to  show  the  antagonism  of  the  com- 
binations and  not  the  actual  growth  of  the  yeast  as  has  been  shown 
in  the  previous  curves. 


98 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.  3 


| 

32 
SO 

— ( 

/ 

Fni  v  fi„m 

i 

MgCl2  X  CaCl 

26 

/,< 

\ 

■ '    -            MgUIo  a  JNaUJ 
KC1  X  MgClo 

\  < 

\\ 

\ 

"""""'" ' ■■    CaCl2  x  NaCl 

\ 

/ 

\ 

/ 

\ 

i 

/ 

\ 

\ 

\  i 

c 
in 

, 

/ 

,  \ 

/ 

\ 

\\ 

/ 

i 

\ 

• 

% 

i 
/ 

i 

\ 

1     '  c 

r> 

fv, 

i 

< 

\ 

\ 

! 

7 

\ 

K 

) — < 

A 

\\ 
\\\ 

\ 

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i 

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w  \ 

i 

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4t 

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1 

4 

;          < 

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// 

1 

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F         G        H         I  J         K        L 

Combinations  of  Salts 


M         N 


Fig.  12. — Curves  showing  range  of  antagonism  of  binary  combinations  of 
salts.  The  ordinates  represent  the  average  number  of  yeast  cells  in  millions 
and  the  abscissae,  the  concentration  of  salts  in  combinations.  The  ordinate  at 
A  represents  the  average  number  of  cells  in  blank  cultures. 


1917]        Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast  99 


Summary 

PART  A— TOXIC  EFFECTS  OF  SINGLE  SALTS 

1.  Each  of  the  four  single  salts— KC1,  MgCl2,  CaCl2,  and  Nad- 
is  more  or  less  toxic  to  the  yeast,  Saccharomyces  ellipsoideus,  at  certain 
concentration.  KC1  is  the  least  toxic  and  NaCl  the  most  for  the  yeast 
(no.  66)  used. 

2.  The  lower  concentrations  of  each  salt  stimulate  the  growth  of 
yeast.  The  highest  number  of  yeast  cells  in  microscopical  count  was 
found  at  .2M  KC1,  .1M  MgCL,  .01M  CaCL,  and  .001M  NaCl,  KC1 
being  the  most  favorable  and  NaCl  the  least.  Beyond  the  favorable 
concentrations  the  various  salts  are  toxic  to  yeast. 

3.  The  concentrations  of  salts  that  inhibited  the  growth  of  yeast 
cells  were  found  at  2.2M  KC1,  1.2M  MgCL,  .7M  CaCl2,  and  .2M  NaCl. 

4.  The  results  of  the  experiments  are  quite  different  from  those 
found  with  either  bacteria,  the  higher  plants  or  animals.  The  yeast 
stands  in  this  respect  midway  between  plants  and  animals  and  swings 
to  either  direction  according  to  the  environment. 

5.  The  salts  used  had  a  marked  effect  on  the  size  and  appearance 
of  the  yeast.  Taking  decrease  in  size  as  a  criterion,  the  salts  affected 
the  yeast  toxically  in  the  same  relative  ways  as  indicated  by  the  rate 
of  multiplication  of  the  cells. 


PART  B— ANTAGONISTIC  EFFECTS  OF  COMBINATIONS  OF  SALTS 

1.  As  shown  by  growth  of  yeast,  the  variation  in  antagonism  be- 
tween the  four  single  salts  in  all  possible  combinations  may  be  ar- 
ranged in  order  as  follows : 

1.  MgCL  vs.  CaCL  (most) 

2.  KC1  vs.  CaCL 

3.  MgCL  vs.  NaCl 

4.  KClvs.  NaCl 

5.  KClvs.  MgCL 

6.  CaCL  vs.  NaCl  (least) 

2.  The  effect  of  binary  salts  with  yeast,  whether  positively  or  nega- 
tively antagonistic  in  comparison  to  animals,  plants  and  soil  bacteria, 
may  be  tabulated  as  follows: 


Yeast 

Animals 

Plants 

Soil  bacteria 

+ 

+  and  - 

+ 

— 

+  and  - 

+  and  — 

+ 

+ 

+ 

+  and  — 

+  and  — 

+ 

+  and- 

+ 

•4-  and  — 

+  and  - 

+  and  — 

+ 
+ 

+ 
+ 

+  and  — 

— 

:  mild  antago 

nism.    —  — 

strong  increase 

of  toxicity.    —  — 

slight 

100  University  of  California  Publications  in  Agricultural  Sciences         [Vol.  3 

Binary  salts 

1.  MgCL  vs.  CaCL 

2.  KC1  vs.  CaCL 

3.  MgCL  vs.  NaCl 

4.  KClvs.  NaCl 

5.  KC1  vs.  MgCL 

6.  CaCL  vs.  NaCl 

-f-  —  strong  antagonism, 
increase  of  toxicity. 

3.  In  regard  to  the  effects  of  valences  of  ions  the  following  results 
have  been  obtained  with  yeast : 

(a)  That  divalent  ions  may  antagonize  monovalent  ions  is  evident  from  the 
combinations  of  MgCl2  vs.  NaCl  and  CaCL  vs.  NaCl.  Ngative  results  were  ob- 
tained from  the  combinations  of  KC1  vs.  CaCL  and  KC1  vs.  MgCl2. 

(b)  That  a  divalent  ion  may  be  antagonized  by  a  divalent  ion  is  evident 
from  the  combination  of  MgCL  vs.  CaCl2. 

(c)  That  monovalent  ions  may  antagonize  divalent  ions  is  shown  in  the 
combinations  of  KC1  vs.  CaCl2;  MgCL  vs.  NaCl  and  KC1  vs.  MgCL. 

(d)  That  a  monovalent  ion  may  antagonize  a  monovalent  ion,  though  not 
very  markedly,  has  been  found  in  the  combination  of  KC1  vs.  NaCl. 


1917]         Mitra:  Toxic  and  Antagonistic  Effects  of  Salts  on  Wine  Yeast  101 


Literature  Cited 

PAET  A— TOXIC  EFFECTS  OF  SINGLE  SALTS 

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